This work has been funded by the National Research Foundation grant NRF-CRP4-2008-02 (J.T.) and Tier 1 grant RG 51/13.

该协议是基于以下资源，这是被称为详细信息，并特别注意事项3，25-28。 洗涤剂胶束2小时 2 O 1.密度匹配注意：该缓冲溶液的密度必须匹配于去污剂胶束的密度。常见的密度调节剂包括2 H 2 O，H 2 18 O，2 H 2 18 O，甘油和蔗糖29。的H 2 18 O具有相同的密度为2的H 2 O和可能是一个更好的选择，如果不希望在该蛋白质可交换质子的氘。在此过程中，将3-（N，N-二dimethylmyristylammonio）-propanesulfonate（C14SB）洗涤剂在50mM Tris pH值7.3的密度，100mM NaCl的将被用2 H 2 O匹配作为初始猜测以下浓度2小时 2 O将被使用：10，30，和50％体积/体积。 1.1。样品制备<ol><li>制备下列储备溶液并过滤消毒用0.2μm注射器过滤：50毫升500毫摩尔Tris pH值7.3和1M NaCl的（10X缓冲液）; 1毫升250毫C14SB（50X洗涤剂溶液）。 </li><li>通过混合20微升10X缓冲液，4微升50X洗涤剂溶液，20微升2 H 2 O（99.9％），和156微升制备的200μl 样品溶液的去离子H 2 O.准备也将200μl的参考溶液通过混合20微升10X缓冲液，20微升2 H 2 O（99.9％），以及160微升的去离子H 2 O. </li><li>对于其他2小时 2 O浓度重复步骤1.1.2，即30％和50％，适当调整2小时 2 O和H 2 O数量。 </li></ol> 1.2。 6通道A大会UC的细胞和样品装载到细胞中。 注意：有两种类型的AUC​​细胞取决于样品装载方法。细胞没有外部填充已被密封之前的细胞加载，而外部填充的细胞被密封后的细胞可以被加载。外部填充的AUC电池组件已经预先3所述。在这个协议中，6声道的AUC细胞没有外部填充的组装进行说明。的主要区别在于，它具有在两侧需要被单独地紧固的螺钉环，并且它不需要壳体插头（图1）。在装配步骤的差以下突出显示。 <img alt="图1" src="/files/ftp_upload/52404/52404fig1.jpg" /> 图1.分解图，无需外部填充一个6通道AUC细胞，这个数字已经被修改Beckman Coulter公司安钛50和AN-60钛AnalytiCAL转子，细胞和动态平衡的用户手册。 <ol><li>准备两个窗口组件蓝宝石窗口 ，而不是石英窗口（图2），每个单元AUC。放置窗口垫圈到窗口保持架。稍微弯曲的窗口衬，并将其放置成使得该间隙形成相对于窗口支架键槽窗口保持架。放置蓝宝石窗口的窗口衬套内部，与窗架键槽对准标记。 注：该石英窗是可压缩的，因此将产生更多的光折射在高速28,30因此对于干扰测量以上30000转，如在此密度匹配实验中，蓝宝石窗口被使用。蓝宝石窗口比石英窗口重并有一个“X”，蚀刻到其一侧。 </li></ol><img alt="图2" src="/files/ftp_upload/52404/52404fig2.jpg" /> 图2.分解图Øf显示窗组件，这个数字已经被修改Beckman Coulter公司安钛50和AN-60钛转子分析，细胞和动态平衡用户手册<ol><li>放置在电池外壳与部件编号颠倒 。用与外壳键对准键槽，滑入电池壳体首先在6个扇区核心与斜口朝下，接着是一个视窗总成朝下的窗口（1图，左）。 </li><li>轻轻大衣螺纹环螺纹和螺纹环垫圈spinkote。放置在窗组件的顶部上的螺纹环垫圈。安装螺纹环到窗口外壳词“OUT”朝外。通过使用电池对准工具手工拧紧螺丝环。 </li><li>使用扭矩扳手，拧紧螺丝环只有60英寸磅 。 </li><li>将与部件号直立放置在12中午细胞。 LOAD 120微升参考到左行和110微升样品进入右行。确保每个样品和参比被正确配对。 注意：精确的样品量不是关键的，但参考需要具有比样品（5-10微升）略多卷，使样品液面将是不同的。 </li><li>小心滑入电池外壳1的窗口组件与窗口朝下（图1，右图）。小心不要过分干扰细胞和溢出的内容。 </li><li> 重复步骤1.2 3和1.2。4，收紧第二螺杆环至120英寸-磅 。倒置细胞并重新拧紧所述第一螺纹环至120英寸-磅 。 </li><li>加载细胞进入转子，所述转子安装到离心机，并根据制造商的说明28安装在单色。 注：本ST详细EP也可以在该参考文献3中找到。 </li></ol> 1.3。设置干扰测量<ol><li>启动用于AUC仪器的用户界面的软件，并在3000rpm下进行激光设置和径向校准对于每个小区，根据制造商的说明，这将简要地总结为以下步骤。 </li><li> 1.3.1.1足够的真空后已经达到（&lt;100微米），以3000rpm运行的离心机。预览在用户接口软件的干涉图案，并调整激光参数，以获得最高的对比度。 </li><li>创建一个新的设置文件（文件|新建文件）指定“平衡”和“干扰”的测量。成立了沉降平衡法（“方法”按钮），在45000转或预期的蛋白质样品的最高速度，以较高者为准，在20℃运行，具有运行温度，并收集1扫描每15分钟一班。生产过程监控通过在HeteroAnalysis打开的数据文件和至少12小时后选择“匹配”函数r平衡进展（大约过夜，图3）。 注：类似的功能也是SEDFIT（选项|加载选项|测试接近平衡）可用。 </li></ol><img alt="图3" src="/files/ftp_upload/52404/52404fig3.jpg" /> 图从HeteroAnalysis匹配功能3.结果的匹配功能可以通过连续扫描和上次扫描之间的比较RMSD监视均衡的进展。这个例子显示了实现平衡8小时后RMSD值渐近于X轴表示。 1.4。数据分析<ol><li>对于每一个组样品，画出对2小时 2 O浓度的径向分布曲线的斜率。 注意：分配将是一个很浅的指数即APproaches线性。 X轴截距对应于匹配的2小时 2 O浓度。 </li><li>为了更准确的结果，进行实验的几个重复。可替换地，重复实验具有较窄范围的2小时 2 O浓度。 </li></ol> SH在C14SB胶束2.沉降平衡 2.1。运行参数<ol><li>通过使用SEDNTERP计算缓冲器的密度和粘度，蛋白质部分比容和离心速度。计算缓冲器的密度和粘度，选择计算中的“缓冲器数据选择”部分，并相应地输入缓冲组件，包括D 2 O中的浓度。 </li><li> 2.1.1.1为了计算蛋白质部分比容，选择计算中的“V-栏”部分，并输入该蛋白质的氨基酸序列。指定“做低聚物从这个单体：N =”最高预期低聚大小字段，在这种情况下，N = 5由在主窗口在RPM字段输入值计算的速度，直到σ≈1;这是一个经验法则，以确保径向分布轮廓25的一个良好的指数形状。 注意：计算该实验的值如下：ρ=1.03839克/ ml时，η= 1.0267厘泊=0.7569毫升/克，ω1 = 16000转。 </li><li>随后的计算速度跟随，以确保在同一速度和未来25分布曲线之间有足够的差异。 注意：这也可以从功能完成“估计平衡转子转速功能”在SEDFIT，其中考虑到了解决方案列（灌装量）。 </li></ol> 2.2。样品制备<ol><li>准备用5毫米C14SB1毫升对照品溶液和32.3％，2小时 2 O从密度匹配实验（第1节），决定将100微升10X缓冲solutioN（步骤1.1.1），20微升50X洗涤剂溶液（步骤1.1.1），323微升2 的 H 2 O（99.9％）和527微升的去离子H 2 O. </li><li>溶解冻干，高效液相色谱法纯化SH肽（表达和纯化先前31描述的）在适当的溶剂如甲醇或50％体积/体积乙腈水溶液。测量溶解的肽的A280在微升尺度的UV / Vis分光光度计和等分对于三个样品以得到A 280，每个时稀释至12mm = 0.3，0.5和0.8（A 280为10mm = 0.25，0.417和0.67） 130微升。冻干样品过夜，并重新悬浮于130微升的参考溶液（步骤2.2.1），得到样品溶液。 注意：SH蛋白可以从紫外/可见光吸收检测到在280nm处，因为它包含的Trp和Tyr残基。蛋白没有芳香族残基可以通过标记它们与合适的生色团，用含色氨酸突变体进行检测，或通过使用干扰NCE测量，而不是吸收。 </li><li>按照第1.2节的步骤来组装一个6通道AUC细胞与石英窗 。加载最高浓度样品（A 280，12毫米 = 0.8）在渠道最接近的转子中心和最低浓度的样品（A 280，12毫米 = 0.3），最远的从转子中心。 </li></ol> 2.3。设置测量吸光度<ol><li>创建一个新的设置文件（文件|新建文件）指定“平衡”和“吸收”的测量。指定280纳米的检测器波长。 </li><li>在3000rpm下通过检查“径向校准第一扫描之前”，在扫描选项，指定数据收集在分辨率低，例如，通过径向步长= 0.01厘米，重复= 3（分辨率低，速度快），并执行单次扫描执行径向校准。扫描完成后，取消选中该选项。 </li><li>成立了沉降平衡法（“其实现方法具D“按钮），以在步骤2.1.3中计算出的第一速度运行，在20℃，并收集1次扫描，每30分钟。在每一个细胞的“详细信息”，在指定低分辨率的数据收集，如步骤2.3.2。至少18小时后在HeteroAnalysis打开数据文件并选择“匹配”功能监视均衡的进展（约一夜之间，图4）。 注意：达到平衡可能需要相当长的时间的时间为第一速度，而随后的速度将花费更少的时间。 </li></ol><img alt="图4" src="/files/ftp_upload/52404/52404fig4.jpg" /> 图4. HeteroAnalysis匹配功能的结果，第一和第二速度（上左和右）似乎已经达到平衡，但最好是等待几个小时，以确保万无一失。相比较而言，在第三和第四速度（底部左侧和右侧）都明显地达到平衡在较短的时间。 <a href="https://www.jove.com/files/ftp_upload/52404/52404fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol><li>一旦达到平衡，收集在高分辨率，例如，使用径向步长= 0.001厘米，重复= 10（高分辨率，慢）的单次扫描。 </li><li>扫描完成后，重复步骤2.3.3和2.3.4下一个速度。 </li><li>任选地，当所需要的时间，以达到平衡的每个速度是已知的（计算或根据经验），建立了沉积平衡法扫描，以包括所有在步骤2.1.3和计算出的速度的平衡时间为每个后收集1次扫描速度。在这种情况下，在每个细胞的“详细信息”指定的高分辨率数据收集。 </li></ol> 2.4。在SEDFIT和SEDPHAT数据分析注：有关详细信息，并考虑在数据分析读者参考吨Ø以下网站：www.analyticalultracentrifugation.com。 <ol><li>在SEDFIT开放高分辨率扫描（数据|负载沉降平衡数据）和分割数据转换成3个通道（这些对应于不同浓度的每种样品;选项|加载选项|减6声道原始数据在3亚群）。 </li><li>重新打开属于在SEDFIT相同样品和相同浓度但不同速度的数据文件。调整半月板（垂直红线），电池底部（垂直蓝线）及配件的限制（垂直绿线），并在SEDPHAT导出数据的使用（数据|导出数据到SEDPHAT）。输入在步骤2.1.1按要求计算，以及转子型和核心类型的参数。重复为每个样品和浓度这一步骤。 </li><li>在SEDPHAT打开从同一样品的所有数据（所有的浓度和速度），并填写实验参数;一个例子示于图。 5。 注：当D 2 O缓冲区中，申加<html>可交换的质子的关合作可以显著改变蛋白质的分子量，特别是对于水溶性蛋白质。膜蛋白，尤其是小的像SH的蛋白质，受到的影响较小，因为膜嵌入式地区免受交流。为了解决这个问题，输入“缓冲液D摩尔分数”。 注：在这一步，建议单独保存编辑后的数据集，通过选择菜单数据|复制所有数据，并另存为新的配置。 </li><li>选择一个模式，并填写该模型的全局参数。 注意：作为一个例子，在“单体 - 正Mer的自协”模型和它的参数示于图。 6。 </li></ol><img alt="图5" src="/files/ftp_upload/52404/52404fig5.jpg" /> 图5.如何填写实验参数的一个例子。 文件/ ftp_upload / 52404 / 52404fig6.jpg“/&gt; 图6.如何填写全局参数为单体-N-Mer的自我关联模型的一个例子。 <ol><li>运行一个全球飞度通过选择菜单飞度|全球飞度，等待合适的收敛。记下（或采取截图）的拟合结果，尤其是在全球减少卡方和日志K A值。提取其他数据，如数据拟合，并从菜单中的复制和显示拟合残差|显示热力学信息。 </li><li>返回到全局参数，并检查M（1）以适合单体分子量和重复步骤2.4.5。记下拟合分子量和全球减少卡方。 </li><li>重复步骤2.4.3至2.4.6每个模型进行测试和比较减少了全球卡方值和拟合残差比较每个模型的拟合质量。 注：小和随机拟合残差通常表示一个不错的选择，而且最适合将模型拥有全球最小的降低卡方。拟合单体分子量和它的卡方值不应该从该固定的（理论）分子量的显着不同。 </li><li>首先选择统计计算所值的置信区间为获得数家|关键卡方错误表面预测和输入所需的置信区间。接下来，进入统计|生成1维误差体表投影，并取消选择登录嘉在全局参数对话框，以获得日志嘉卡方值。 注：建议读者咨询更多详细信息如下源（http://www.analyticalultracentrifugation.com/ sedphat / statistics.htm）这种方法33的方法32，以及插图。 </li></ol>

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The authors have nothing to disclose.

本文提供了一个实验方案样品制备和使用均衡沉降的小膜蛋白的洗涤剂齐聚分析。所描述的协议是同样有效-and simpler-为可溶性蛋白，作为密度匹配步骤是不需要的。实际上，该系统是由洗涤剂和蛋白质的混合​​物构成。进行沉降研究，该洗涤剂必须是不可见的引力场，使得它不向粒子浮选。因此，洗涤剂的密度，必须仔细地通过加入D 2 O中的到缓冲匹配，与限制，即如果所使用的洗涤?...

分析超速离心1-5是研究大分子相互作用的生理条件下，被访问既弱相互作用和强相互作用中最重要的方法之一。该方法是无标记，并使用光的吸收或干扰，并且甚至荧光光学系统可用于多级6几个数量访问浓度范围。 因为大多数生化过程取决于可逆的相互作用，此方法特别有用。这些相互作用的化学计量和强度必须被定量表征理解生物过程，以及用于此目的7,8许多方法存在的。然而，瞬时相互作用是难以研究9。 一个方法的选择来表征大分子相互作用取决于它的静态或动态性质。在第一种情况下，sedim<html> entation速度（SV）的情况下，其中的径向传输的速率测量和复合物分馏的浮力质量和形状的差异的基础上。 与此相反，动态连接是可逆的实验的时间尺度，不能物理地分离。在这种情况下，自或杂相互作用导致的非共价相互作用以依赖于总蛋白浓度的平衡。这些动态的相互作用可以通过两个沉降平衡（SE）和沉降速度（SV）10进行研究。然而，第一种方法是简单地履行和这里进行说明。在SE，离心是在足够低的速度进行，使得扩散和沉淀之间达到平衡。在这一点上，一个光信号（UV-VIS）的作为径向距离的函数的平衡轮廓，可以使用预先设定的热力学模型协会11进行分析。 ve_content“&gt;在本文件中，一个沉降平衡研究，提出一种病毒的膜蛋白，其形成由于其疏水性的离子通道。的自缔合的，实验运行在洗涤剂的存在下，以及在这种情况下，密度溶剂具有被匹配到该洗涤剂的，但是，该协议描述中的水溶性蛋白的情况下将是相同的，所不同的是将需要无溶剂的密度匹配。 使用的蛋白质被编码在人呼吸道合胞病毒（hRSV的），在副粘病毒科，导致下呼吸道疾病的婴幼儿，老年人和免疫受损人群全世界12有包膜肺病毒。每年高达6400万报hRSV的感染病例和16万死亡病例发生。 的hRSV的基因组转录11蛋白，包括三个膜蛋白F，G和小疏水性（SH）。 SH蛋白参与在RSV感染的发病机制。 RSV缺乏的SH基因（RSVΔSH）是可行的，导致形成合胞体和生长以及野生型（WT）病毒13-16。然而，RSVΔSH病毒复制在上呼吸道15,16 10倍效率较低比WT上。另外，RSVΔSH病毒减毒在体内小鼠和黑猩猩模型13,17。 该SH蛋白是64（RSV亚组A）或65（RSV亚组B）氨基酸长II型整合膜蛋白的积累主要是在高尔基体18的膜。 SH蛋白具有单个预测α-螺旋跨膜（TM）结构域19，其高度保守20,21。的C-和N-末端膜外结构域被定向lumenally /细胞外和细胞质，分别。 两个合成TM结构域（残基18-43）和全长的SH蛋白已经显示出，以形成在各种洗涤剂homopentamers。该homopentameric形式是负责平面脂质双层22,23通道活动。首先确定使用位点特异性的红外二色性23，其显示的His-22是在一个腔，靠近跨螺旋，取向在脂双层对TM单体的正确方向。相同的TM域取向用NMR研究，重建了五聚体的全长蛋白质的α-螺旋束中dodecylphosphocholine（DPC）胶束22证实。在这种“胶团”的模式中，单个的α-螺旋TM结构域侧翼的N-末端由一个螺旋，和C末端由一个扩展B-发夹。 SH蛋白的两个质子化的残基，的His-22和的His-51，分别位于在TM结构域（lumenally取向），并且在膜外C末端β发夹的尖端（远离通道孔），分别为。在一个bicellar ENVIRO的联邦政府，但是，对TMα螺旋延伸到的His-51，并且两个His残基可访问的信道24的内腔中。该信道结构采用漏斗状结构22，其中，窄的区域（丝氨酸29至Cys-45）22衬有疏水性侧链（岛-32，岛-36，岛-40和Leu-44），并ILE-36限定在通道管腔最窄处。他-22位于该漏斗的最大开口，而他的-51是在最小的开口的前端。 在本文件中，在一个沉降平衡模式分析离心已被用来确定是否他质子影响的SH蛋白五聚体的稳定性。在这种情况下，SH蛋白溶解于C 14甜菜碱洗涤剂，其先前已用于显示的SH蛋白形式的五聚体的低聚物22。

<table border="0" cellpadding="0" cellspacing="0" width="1345">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:448px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">3-(N,N-dimethylmyristylammonio)propanesulfonate</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:119px;">T0807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Deuterium oxide 99.8%</td>
			<td style="width:147px;">Cambridge Isotope</td>
			<td style="width:119px;">DLM-4-99.8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">An-50 Ti Rotor, Analytical, 8-Place</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">363782</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">An-60 Ti Rotor, Analytical, 4-Place</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">361964</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Cell housing</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">334784</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">12 mm six-channel centerpiece, epon charcoal-filled</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">331376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Window holder</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">305037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Window gasket</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">327021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Window liner</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">362329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Sapphire window</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">307177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Quartz window</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">301730</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Screw-ring washer</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">362328</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Screw ring</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">301922</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Spinkote</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">306812</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Torque stand assembly</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">361318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Counterbalance</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">360219</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Cell alignment tool</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">362340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SEDNTERP</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td>http://bitcwiki.sr.unh.edu/index.php/Main_Page</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HeteroAnalysis&#160;</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td>http://www.biotech.uconn.edu/auf/?i=aufftp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SEDFIT</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td>http://www.analyticalultracentrifugation 
			.com/sedfit.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SEDPHAT</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td>http://www.analyticalultracentrifugation 
			.com/sedphat/default.htm</td>
		</tr>
	</tbody>
</table>

sedimentation equilibrium of a small oligomer-forming membrane protein: effect of histidine protonation on pentameric stability

分析超速离心（AUC），可用于研究大分子之间可逆的相互作用在宽范围的相互作用的优势和在生理条件下。这使得AUC选择的方法，以定量评估化学计量学和同和异的关联是短暂的，可逆的生化过程的热力学。在沉降平衡的模态（SE），扩散和沉淀之间的平衡提供了一种简档作为径向距离的函数的依赖于特定的关联模型。这里，详细的SE协议被描述来确定一个小的膜蛋白低聚物，使用分析超速离心机的大小和单体 - 单体关联的能量。 AUC-ES是无标记，仅根据物理原理，并能在两个水溶性和膜蛋白可以使用。一个例子示出了后者的，小疏水（SH）在人呼吸道合胞病毒的蛋白质（hRSV的），一个65个氨基酸的多肽与单个α螺旋跨膜（TM）结构域，其形成五聚体离子通道。基于NMR的结构数据显示，SH蛋白具有在其跨膜结构域2质子化His残基被定向朝向所述通道的内腔。 SE试验的目的是为了确定如何pH值影响缔合常数和SH蛋白的寡聚大小。而五聚体形式在所有的情况下保存，其缔合常数降低在低pH值。这些数据与观察SH通道活性类似的pH依赖性，在SH蛋白两种氨酸残基的腔的方向一致的协议。后者可在低pH值会遇到的静电斥力和降低的低聚物的稳定性。总之，这种方法适用于每当在生理条件微妙的蛋白质 - 蛋白质关联的变化的定量信息必须被测量。

的C14SB去污剂胶束的径向分布曲线在50mM Tris，100mM NaCl的pH为7.3的形式，可以被安装到一个线性模型（ 图7A）一个很浅的指数。这种分布的斜率成反比关系到D 2 O中的浓度（ 图7B）。的点的斜率是零， 也就是，匹配D 2 O中的浓度，结果为32.3％。 参见图7所示。 相同的实验重复进行不同...

Watch this Scientific Journal Video about Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability at JoVE.com

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).

小型低聚物形成膜蛋白的沉降平衡：组氨酸质子对五聚体稳定性

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and ...

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10.5K 次观看.  Nanyang Technological University. Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).

视频: Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

铱（III）荧光探针检测疟疾蛋白质生物标志物组氨酸丰富的蛋白质-Ⅱ

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

科研

化学

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

氯离子的动态电化学测试

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

定点自旋标记和五聚体配体门控离子通道的EPR光谱研究

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

Original Experimental Approach for Assessing Transport Fuel Stability

The study of fuel oxidation stability is an important issue for the development of future fuels. Diesel and kerosene fuel systems have undergone several technological changes to fulfill environmental and economic requirements. These developments have resulted in increasingly severe operating conditions whose suitability for conventional and alternative fuels needs to be addressed. For example, fatty acid methyl esters (FAMEs) introduced as biodiesel are more prone to oxidation and may lead to deposit formation. Although several methods exist to evaluate fuel stability (induction period, peroxides, acids, and insolubles), no technique allows one to monitor the real-time oxidation mechanism and to measure the formation of oxidation intermediates that may lead to deposit formation. In this article, we developed an advanced oxidation procedure (AOP) based on two existing reactors. This procedure allows the simulation of different oxidation conditions and the monitoring of the oxidation progress by the means of macroscopic parameters, such as total acid number (TAN) and advanced analytical methods like gas chromatography coupled to mass spectrometry (GC-MS) and Fourier Transform Infrared - Attenuated Total Reflection (FTIR-ATR). We successfully applied AOP to gain an in-depth understanding of the oxidation kinetics of a model molecule (methyl oleate) and commercial diesel and biodiesel fuels. These developments represent a key strategy for fuel quality monitoring during logistics and on-board utilization.

Oxidation stability of transport fuels has become a concern for future fuel development. This work presents an original methodology developed by IFP Energies Nouvelles for assessing fuel stability using two different reactors. This methodology was successfully applied to gain an in-depth understanding of the oxidation kinetics and pathways of model molecules and commercial fuels.

评定运输燃料的稳定性原始实验方法

The study of fuel oxidation stability is an important issue for the development of future fuels. Diesel and kerosene fuel systems have undergone several ...

Non-equilibrium Microwave Plasma for Efficient High Temperature Chemistry

A flowing microwave plasma based methodology for converting electric energy into internal and/or translational modes of stable molecules with the purpose of efficiently driving non-equilibrium chemistry is discussed. The advantage of a flowing plasma reactor is that continuous chemical processes can be driven with the flexibility of startup times in the seconds timescale. The plasma approach is generically suitable for conversion/activation of stable molecules such as CO2, N2 and CH4. Here the reduction of CO2 to CO is used as a model system: the complementary diagnostics illustrate how a baseline thermodynamic equilibrium conversion can be exceeded by the intrinsic non-equilibrium from high vibrational excitation. Laser (Rayleigh) scattering is used to measure the reactor temperature and Fourier Transform Infrared Spectroscopy (FTIR) to characterize in situ internal (vibrational) excitation as well as the effluent composition to monitor conversion and selectivity.

This article describes a flowing microwave reactor that is used to drive efficient non-equilibrium chemistry for the application of conversion/activation of stable molecules such as CO2, N2 and CH4. The goal of the procedure described here is to measure the in situ gas temperature and gas conversion.

用于高效高温化学的非平衡微波等离子体

A flowing microwave plasma based methodology for converting electric energy into internal and/or translational modes of stable molecules with the purpose ...

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the average content of fluorinated drugs in their formulated drug product forms: tablets or capsules. This method is specific to fluorinated drugs because it detects only the content of fluorine, avoiding interference from the excipients that lack fluorine. The advantages of measuring the active content of fluorinated drugs using low-field 19F TD-NMR versus high-field 19F solid-state (SS) NMR are the simplicity of the method; the low cost; and the non-destructive nature of the technique, with all samples recoverable in intact forms (e.g., powders, tablets, and capsules), making this technique affordable for any laboratory.
We have tested the method with three fluorinated drug products available on the market - cinacalcet, lansoprazole, and ciprofloxacin - with doses ranging from 15 to 500 mg. The results of the analyses, measured by low-field 19F TD-NMR, supported the reported label claims for the average drug content.
Based on the simplicity and reproducibility of the analysis, we envision this methodology being implemented in any laboratory, including manufacturing plants, as a process analytical technology (PAT) tool in the pharmaceutical industry.

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

A simple and non-destructive technique that measures the average content of drug substances in formulated drug products containing fluorine using low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) is presented here. The technique can be applied to the development and manufacturing of drugs in the pharmaceutical industry.

应用程序和非破坏性19F 时间域核磁共振技术的方法来衡量中含氟药物产品的内容

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the ...

The Effect of Interfacial Chemical Bonding in TiO2-SiO2 Composites on Their Photocatalytic NOx Abatement Performance

The chemical bonding of particulate photocatalysts to supporting material surfaces is of great importance in engineering more efficient and practical photocatalytic structures. However, the influence of such chemical bonding on the optical and surface properties of the photocatalyst and thus its photocatalytic activity/reaction selectivity behavior has not been systematically studied. In this investigation, TiO2 has been supported on the surface of SiO2 by means of two different methods: (i) by the in situ formation of TiO2 in the presence of sand quartz via a sol-gel method employing tetrabutyl orthotitanium (TBOT); and (ii) by binding the commercial TiO2 powder to quartz on a surface silica gel layer formed from the reaction of quartz with tetraethylorthosilicate (TEOS). For comparison, TiO2 nanoparticles were also deposited on the surfaces of a more reactive SiO2 prepared by a hydrolysis-controlled sol-gel technique as well as through a sol-gel route from TiO2 and SiO2 precursors. The combination of TiO2 and SiO2, through interfacial Ti-O-Si bonds, was confirmed by FTIR spectroscopy and the photocatalytic activities of the obtained composites were tested for photocatalytic degradation of NO according to the ISO standard method (ISO 22197&#8722;1). The electron microscope images of the obtained materials showed that variable photocatalyst coverage of the support surface can successfully be achieved but the photocatalytic activity towards NO removal was found to be affected by the preparation method and the nitrate selectivity is adversely affected by Ti-O-Si bonding.

The Effect of Interfacial Chemical Bonding in TiO2-SiO2 Composites on Their Photocatalytic NOx Abatement Performance

The focus of the present work is to establish means to generate and quantify levels of Ti-O-Si linkages and to correlate these with the photocatalytic properties of the supported TiO2.

界面化学键合在TiO中的作用<sub&gt; 2</sub&gt; -SiO<sub&gt; 2</sub&gt;复合材料对其光催化NOx消除性能的影响

The chemical bonding of particulate photocatalysts to supporting material surfaces is of great importance in engineering more efficient and practical ...

The Effect of Charging and Discharging Lithium Iron Phosphate-graphite Cells at Different Temperatures on Degradation

The effect of charging and discharging lithium iron phosphate-graphite cells at different temperatures on their degradation is evaluated systematically. The degradation of the cells is assessed by using 10 charging and discharging temperature permutations ranging from -20 &#176;C to 30 &#176;C. This allows an analysis of the effect of charge and discharge temperatures on aging, and their associations. A total of 100 charge/discharge cycles were carried out. Every 25 cycles a reference cycle was performed to assess the reversible and irreversible capacity degradation. A multi-factor analysis of variance was used, and the experimental results were fitted showing: i) a quadratic relationship between the rate of degradation and the temperature of charge, ii) a linear relationship with the temperature of discharge, and iii) a correlation between the temperature of charge and discharge. It was found that the temperature combination for charging at +30 &#176;C and discharging at -5 &#176;C led to the highest rate of degradation. On the other hand, the cycling in a temperature range from -20 &#176;C to 15 &#176;C (with various combinations of temperatures of charge and discharge), led to a much lower degradation. Additionally, when the temperature of charge is 15 &#176;C, it was found that the degradation rate is nondependent on the temperature of discharge.

This article describes the effect of dissimilar charging/discharging temperatures on the degradation of lithium iron phosphate-graphite pouch cells, aiming at simulating close to real case scenarios. In total, 10 temperature combinations are investigated in the range -20 to 30 &#176;C in order to analyze the impact of temperature on degradation.

不同温度下的磷酸铁锂-石墨电池充放电对降解的影响

The effect of charging and discharging lithium iron phosphate-graphite cells at different temperatures on their degradation is evaluated systematically. ...

The Effect of Ultraviolet Radiation on the Chemical Bath Deposition of Bis(thiourea) Cadmium Chloride Crystals and the Subsequent CdS Obtention

In this work, the effects on the preparation of bis(thiourea) cadmium chloride crystals when illuminated with ultraviolet (UV) light at a wavelength of 367 nm using the chemical bath deposition technique are studied comparatively. Two experiments are performed to make a comparison: one without UV light and the other with the aid of UV light. Both experiments are performed under equal conditions, at a temperature of 343 K and with a pH of 3.2. The precursors used are cadmium chloride (CdCl2) and thiourea [CS(NH2)2], which are dissolved in 50 mL of deionized water with an acidic pH. In this experiment, the interaction of electromagnetic radiation is sought at the moment the chemical reaction is carried out. The results demonstrate the existence of an interaction between the crystals and the UV light; the UV light assistance causes crystal growths in an acicular shape. Also, the final product obtained is cadmium sulfide and shows no evident difference when synthesized with or without the use of UV light.

The Effect of Ultraviolet Radiation on the Chemical Bath Deposition of Bisthiourea Cadmium Chloride Crystals and the Subsequent CdS Obtention

This article presents a protocol for the synthesis of bis(thiourea) cadmium chloride crystals by chemical bath deposition. Two experiments are described: one aided by ultraviolet light compared to one without ultraviolet light.

紫外辐射对双 (硫脲) 氯化镉晶体化学镀液沉积的影响及随后的 CdS 获得

In this work, the effects on the preparation of bis(thiourea) cadmium chloride crystals when illuminated with ultraviolet (UV) light at a wavelength of ...

Niobium Oxide Films Deposited by Reactive Sputtering: Effect of Oxygen Flow Rate

Reactive sputtering is a versatile technique used to form compact films with excellent homogeneity. In addition, it allows easy control over deposition parameters such as gas flow rate that results in changes on composition and thus in the film required properties. In this report, reactive sputtering is used to deposit niobium oxide films. A niobium target is used as metal source and different oxygen flow rates to deposit niobium oxide films. The oxygen flow rate was changed from 3 to 10 sccm. The films deposited under low oxygen flow rates show higher electrical conductivity and provide better perovskite solar cells when used as electron transport layer.

Here, we present a protocol for niobium oxide films deposition by reactive sputtering with different oxygen flow rates for use as an electron transport layer in perovskite solar cells.

活性溅射沉积的氧化氮薄膜：氧气流速效应

Reactive sputtering is a versatile technique used to form compact films with excellent homogeneity. In addition, it allows easy control over deposition ...